Project Summary Herpesviruses are major causes of human infectious diseases. Sexually transmitted herpes simplex virus 2 (HSV-2) afflicts more than 400 million people world-wide, increases acquisition and transmission of HIV, and can be lethal when babies are infected during birth. HSV-1 is the leading cause of infectious blindness and is increasingly spread by sexual contact. Current anti-herpesviral therapies based on nucleos(t)ide analogs such as acyclovir (ACV) are inadequately suppressive in some patients. Nucleoside analogs also readily select for resistance mutations, which are difficult to treat with current second line drugs such as foscarnet due to toxicity. Therefore more effective therapeutic options and/or drugs with a different mechanism of action are urgently needed for use in salvage and combination therapies. Herpesvirus replication relies on several activities catalyzed by nucleotidyl transferase superfamily (NTS) enzymes. We have strong preliminary data that several compounds known to inhibit NTS enzymes profoundly suppress HSV replication in cell culture, some further than ACV even without optimization. Our data also indicate the NTS enzyme inhibitors in the polyoxygenated heterocycle (POH) compound class, including N-hydroxypyridinones (HPOs) and N-hydroxynapthyridinones (HNOs), have a different mechanism of action than ACV and have a higher barrier to development of resistance. Specifically, the viral terminase has been identified as a viral target. Together, our observations suggest NTS inhibitors have strong potential for development into antiviral drugs. We propose to derive preliminary structure-activity relationships (SARs) for the HPOs and HNOs and identify their mechanism(s) of action. This will provide essential information to guide future medicinal chemistry optimization. We will also test the hypothesis that these POH compounds can be useful in combination therapy with the established anti-HSV drugs ACV and foscarnet. These goals will be pursued in two Aims: Aim 1: Test HPO and HNO compounds against HSV replication to develop preliminary SARs. Aim 2: Assess the mechanism of action of HPO and HNO inhibitors and evaluate potential synergy with ACV and foscarnet to suppress HSV replication. Aim 1 will use our well-established, robust screening procedure that tests inhibition of HSV replication by the compounds, toxicity, and in vitro absorption, distribution, metabolism and excretion (ADME) to develop the foundational SARs needed to formulate focused chemical hypotheses to be tested in future medicinal chemical optimization. Aim 2 will investigate the compounds' mechanism of action and use our replication inhibition assay in matrix fashion to test the potential for HPOs and/or HNOs to be useful in combination therapy with each other or with nucleoside analogs or second-line drugs. Follow-up studies will identify lead compounds for development into drugs to improve therapy and relieve suffering due to HSV and other herpesviruses.